Immunization protocols involve the injection of a substance capable of stimulating an immunogenic response into an animal and, after some period of time, bleeding the animal and recovering the antibodies so produced. The immunogenic substance, termed "an antigen," is generally dissolved or suspended in a matrix containing an adjuvant, such as Freund's Adjuvant, alum, etc., to enhance the immune response. Booster shots are also employed after the initial injection in order to enhance the response.
Even with the use of adjuvants and boosters, there are certain substances with potential antigenic characteristics which are simply too small to induce an antibody response when directly injected into an animal. Peptides, mycotoxins or other small molecules, termed "haptens," are examples of such substances. To utilize these as immunogenic substances, it is customary to conjugate, i.e. cross-link, them to a carrier protein such as bovine serum albumin (BSA) prior to injection. Coupling of the hapten to the protein is accomplished by reaction of the hapten with the functional groups of the protein.
As discussed by Chu, et al., in "Journal of Immunological Methods" 55 (1982) 73-78, the amount of hapten conjugated to each mole of carrier protein plays an important role in determining the quantity and specificity of the antibody response to the conjugate. In this instance, a large excess of mycotoxin was found to be necessary in the coupling reaction to yield a conjugate with the appropriate degree of substitution to function as a good antigen.
What Chu, et al., disclosed in the previously cited article is that modification of BSA with ethylene diamine gave an increase in the number of amino groups present on the carrier and thus enhanced the coupling efficiency of the hapten, mycotoxin. In turn, it is disclosed that the use of this resultant conjugate produces substantially higher antibody titers more rapidly than when the unmodified carrier conjugate was used.
Concerning substances which are themselves immunogenic without coupling to a carrier, e.g. BSA, researchers at the University of Cincinnati College of Medicine have published a series of papers reporting on how the response can be improved by cationizing the antigens prior to animal injection. The papers are as follows:
1. Muckerheide, et al., "The Journal of Immunology," 138, 833-837 (1987). PA1 2. Muckerheide, et al., "The Journal of Immunology," 138, 2800-2804 (1987). PA1 3. Domen, et al., "The Journal of Immunology," 139, 3295-3198 (1987). PA1 4. Apple, et al., "The Journal of Immunology," 140, 3290-3295 (1988).
As illustrated in these articles, cationization of BSA is achieved by substituting native anionic side chain carboxyl groups with cationic amino ethylamide groups. As with Chu, this is achieved by reacting BSA with ethylenediamine.
While the articles in the "Journal of Immunology" identified above establish the fact that large molecule like BSA can be made an even more potent immunogen by cationization, to carry this concept over to other antigens may be time consuming and difficult. There are a number of proteins which the scientific community is interested in evaluating for immunogenic characteristics, and the protocol for cationizing each of them to achieve optimum enhancement is unique. In addition, direct cationization of some antigens may destroy or modify their native antigenic determinants, thus rendering them useless.